Collectively areas of research, such as genomics, transcriptomics, and proteomics, or areas of integrated research thereof have been referred to as “omics” or “omics research” in recent years.
The genomics targets genomic DNA. The genomic analysis discovers DNA polymorphisms or factors contributing to genetic disease and reveals the association of disease with such polymorphisms, mutations, or factors.
The transcriptomics targets transcribed RNA. The transcriptomic analysis reveals the correlation of gene expression levels with disease, biological phenomena, or the like.
Furthermore, the proteomics targets proteins. The proteomic analysis offers findings on the association of protein expression levels with disease, biological phenomena, or the like, based on the identification and quantification of particular proteins.
All of these pieces of biological information obtained by genomics, transcriptomics, proteomics, and metabolomics (which is based on metabolites in plasma) are comprehensively analyzed to provide for efficient research. Furthermore, the obtained findings can be used very effectively in basic research or in disease diagnosis or treatment. Thus, a breakthrough in omics research is expected.
Moreover, it has been reported in recent years that the molecular profiles of peripheral blood cells reflect physiological and pathological transformation occurring in various tissues in the body. Along with this, diagnosis has been attempted by the analysis of proteins, metabolites, and gene expression in blood cells.
Meanwhile, for example, the isolation of DNA, RNA, and proteins from, for example, blood cells, involves initially recovering the blood cells and further requires subsequent complicated procedures. DNA targeted by genomic analysis is present in nuclear membranes; RNA targeted by transcriptomic analysis is present in cytoplasmic ribosomes; and proteins targeted by proteomic analysis are present in cell membranes, cytoplasms, nuclear membranes, and so on. Therefore, to prepare DNA, RNA, and proteins from cells, in general, these components are prepared through different routes under the present circumstances. Since the preparation of, for example, nucleic acids (DNA or RNA), targets only these nucleic acids, other components, particularly, proteins, are often denatured with a strong denaturant. Moreover, their respective preparations are complicated. A convenient and efficient extraction method has not been developed yet.
A conventional method for preparing nucleic acids will be described below. In the conventional preparation of nucleic acids, cells are first recovered and then lysed by physical treatment or treatment with a surfactant. Then, impurities are removed therefrom using an organic solvent such as water-saturated phenol or chloroform. In this method, in general, a nucleic acid fraction in the solution is subsequently precipitated with alcohol and further purified, if necessary, by column chromatography (Lectures on Biochemical Experiments 2 (Tokyo Kagaku Dojin), “Nucleic Acid Chemistry I” p. 74-80, p. 262-270, Gene Manipulation Manual (Kodansha Ltd.), p. 20-23, 1982).
In this method, proteins in the cell membranes are often denatured due to the surfactant treatment for lysing the cells. For example, cationic, anionic, nonionic, and amphoteric surfactants are used as the surfactant. On the other hand, proteins in the cytoplasms are also denatured by the phenol or chloroform treatment in the conventional method. This phenol or chloroform treatment requires the procedure of separating an organic solvent layer and a nucleic acid layer by centrifugation or the procedure of performing column chromatography. Moreover, phenol is toxic and causes chemical burn upon contact with the skin. Chloroform is anesthetically active. Therefore, this method also has the disadvantage that the handling or disposal method of these chemicals is an issue.
Furthermore, RNA preparation requires suppressing decomposition, because RNA is exceedingly easily decomposed by RNase. Therefore, a generally adopted method includes denaturing proteins using an RNase inhibitor or a chaotropic agent and then separating an RNA fraction based on adsorption to silica. In fact, many commercially available kits adopt this method. The chaotropic agent generates chaotropic ions (monovalent anions having a large ionic radius) when added to an aqueous solution and has the effect of increasing the water solubility of hydrophobic molecules. Specific examples thereof include alkali iodide, guanidine thiocyanate, alkali metal salts of perchloric acid, alkali metal salts of trifluoroacetic acid, alkali metal salts of trichloroacetic acid, and alkali metal salts of thiocyanic acid. These chaotropic agents in use do not require using organic solvents such as phenol and chloroform and therefore eliminate the problem associated with the handling of phenol or chloroform. However, even when the chaotropic agents are used, the fact remains that proteins are strongly denatured.
Next, DNA and RNA separation will be described. DNA is present in nuclear membranes, and RNA is present in cytoplasms. Thus, cells are treated under conditions that lyse cell membranes but do not lyse nuclear membranes to recover only the nuclei. As a result, DNA and RNA can be separated. As described above, ionic surfactants are used for lysing cell membranes. Of the ionic surfactants, particularly, anionic surfactants lyse nuclear membranes and nucleoproteins and as such, cannot be used in the DNA and RNA separation.
A method for lysing only cell membranes without lysing nuclear membranes is disclosed in New Lectures on Biochemical Experiments 2 (ed., by The Japanese Biochemical Society), Nucleic Acid I, Separation and Purification, p. 49. In this method, cells are treated with NP-40 or Triton X-100 at a final concentration of 0.3%. Furthermore, conditions for nuclear separation described therein involve a Triton X-100 concentration of 0.3% or 1%, an NP40 concentration of 0.1 to 0.5%, and a Tween 20 concentration of 1%.
However, as seen from results of similar experiments conducted by the present inventors, the obtained RNA sample may be contaminated with DNA, and the nuclear membranes are partially dissolved. Particularly, this tendency was apparent for a small number of cells. Therefore, it is difficult for the above method of New Lectures on Biochemical Experiments 2 to obtain a DNA or RNA sample uncontaminated with the other component.
Moreover, Japanese Patent Application Laid-Open No. 2006-51042 discloses a method including directly subjecting cytoplasmic mRNA to RT-PCR, wherein only cell membranes are dissolved without dissolving nuclear membranes to separate cytoplasmic mRNA. Specifically, the disclosed method includes suspending cultured cells for approximately 5 minutes in a solution containing 0.1 to 0.5% NP-40 as a surfactant in 10 mM tris-HCl (pH 7.6) and then centrifuging (1200 g, 5 min) the suspension to recover a nuclear fraction as precipitates. However, this method is merely intended to reduce the amount of contaminating DNA for enhancing RT-PCR efficiency and does not completely separate DNA and RNA.
Disclosures of other nuclear separation methods can be referred to Patent Japanese Patent Application Laid-Open No. H6-205676 and U.S. Pat. No. 6,718,742. Japanese Patent Application Laid-Open No. H6-205676 discloses a method including: adding a buffer solution containing 0.32 M saccharose, 5 mM magnesium chloride, 1% Triton X-100, and 0.2% sodium azide to the whole blood; then recovering nuclei by centrifugation at 12000 rpm for 20 seconds; further lysing nuclear membranes and nucleoproteins by treatment with a surfactant and a protease; and then separating DNA strands by contact with a chaotropic agent.
Moreover, U.S. Pat. No. 6,718,742 discloses that a solution containing 1% Tween 20 in 10 mM ammonium bicarbonate (pH 9.0) is effective for nuclear separation.
As described above, the conventional methods hardly detected, identified, or quantified a nucleic acid and a protein from one sample. Moreover, all of these methods were complicated and did not prepare a nucleic acid or the like conveniently and efficiently from a cell.